BCA Assay Trial 1

Practice makes perfect?

Today we attempted a BCA Assay to quantify the amount of protein in our sample. However, the microplate reader in the Genome Sciences building wasn’t working! We’ll need to repeat this assay on Monday, Dec. 12, but here’s what we did:

The first step in our protocol involved making the necessary reagents.

BCA Working Reagent

Got BCA kit

Pipetted 20 mL of BCA Reagent A into a falcon tube

Added 400 µL BCA Reagent B

Vortexed to mix thoroughly

50mM NH4HCO3 solution

Pipetted 5 mL nanopure water to a falcon tube

Weighed 0.03953g of NH4HCO3 and added to nanopure

Vortexed to mix thoroughly

Poured contents of falcon tube into a graduated cylinder

Pipetted nanopure water into the graduated cylinder until total volume was 10 mL

Poured contents of graduated cylinder back into the falcon tube

Vortexed to mix thoroughly

Lysis Buffer

Pipetted 4 mL of 50mM NH4HCO3 solution into a new falcon tube

Weighed 1.44g Urea and added to falcon tube

Vortexed to mix thoroughly

Poured contents of falcon tube into a graduated cylinder

Pipetted nanopure water into the graduated cylinder until total volume was 6 mL

Poured contents of graduated cylinder back into the falcon tube

Vortexed to mix thoroughly

BCA Standards

Using the table below, we added a designated amount of the Lysis Buffer to a specified solution in snaptop centrifuge tubes. Pulse three times on vortex to mix thoroughly

Table 1. Volume of specified solution and Lysis Buffer added to each vial, along with resulting BSA concentration.

Creating the BCA Assay Microplate

We mapped out where each sample would go on the microplate in the table below, and pipetted accordingly. Each well contained 10 µL of the solutions designated in the table, as well as 200 µL of the BCA working reagent. The total volume in each well was 300 µL.

Table 2. Microplate arrangement.

Well Number

Contents

Replicate

A1

Vial B

1

A2

Vial B

2

A3

Vial B

3

A4

Vial C

1

A5

Vial C

2

A6

Vial C

3

A7

Vial D

1

A8

Vial D

2

A9

Vial D

3

A10

Vial E

1

A11

Vial E

2

A12

Vial E

3

B1

Vial F

1

B2

Vial F

2

B3

Vial F

3

B4

Vial G

1

B5

Vial G

2

B6

Vial G

3

B7

Vial H

1

B8

Vial H

2

B9

Vial H

3

B10

Vial I

1

B11

Vial I

2

B12

Vial I

3

C1

G10

1

C2

G10

2

C3

G10

3

C4

G18

1

C5

G18

2

C6

G18

3

C7

G48

1

C8

G48

2

C9

G48

3

C10

G58

1

C11

G58

2

C12

G58

3

D1

G68

1

D2

G68

2

D3

G68

3

D4

G77

1

D5

G77

2

D6

G77

3

D7

G92

1

D8

G92

2

D9

G92

3

D10

G97

1

D11

G97

2

D12

G97

3

E1

G119

1

E2

G119

2

E3

G119

3

E4

G131

1

E5

G131

2

E6

G131

3

E7

O07

1

E8

O07

2

E9

O07

3

E10

O15

1

E11

O15

2

E12

O15

3

F1

O37

1

F2

O37

2

F3

O37

3

F4

O47

1

F5

O47

2

F6

O47

3

F7

O55

1

F8

O55

2

F9

O55

3

F10

O77

1

F11

O77

2

F12

O77

3

G1

O107

1

G2

O107

2

G3

O107

3

G4

O119

1

G5

O119

2

G6

O119

3

G7

O127

1

G8

O127

2

G9

O127

3

G10

O142

1

G11

O142

2

G12

O142

3

H1

GBLANK

1

H2

GBLANK

2

H3

GBLANK

3

H4

OBLANK

1

H5

OBLANK

2

H6

OBLANK

3

We then covered the microplate and brought it over to the Genome Sciences building (and discovered the plate reader wasn’t working). We’ll have to repeat this entire protocol on Monday!